Pentamidine

Chemical Synthesis of Pentamidine and Analogues

The synthesis of this compound and its derivatives involves strategic organic transformations to achieve the desired diamidine structure. Classical routes and modern adaptations are discussed below, with emphasis on reaction mechanisms, yields, and structural variants.

Classical Synthesis Routes

The foundational synthesis of this compound relies on the coupling of aromatic nitriles with diamidine precursors. A pivotal method involves the Williamson ether synthesis followed by Pinner reaction to introduce amidine groups. For instance, 4-cyanophenol reacts with cis-1,4-dichloro-2-butene to form a dicyano intermediate (10 ). Subsequent Pinner reaction converts the nitriles into imidate esters (11 ), which undergo ammonolysis to yield the bisamidino product (4 ). This route, producing cis-butamidine congeners, demonstrates a yield of 79% under optimized reflux conditions.

Table 1: Key intermediates in this compound synthesis

The trans-isomers (5 , 7 ) are synthesized analogously using trans-1,4-dichloro-2-butene, highlighting the stereochemical flexibility of the synthetic pathway.

Synthesis of Structural Analogues

Modifying this compound’s core structure has yielded analogues with enhanced bioavailability and reduced toxicity. For example, compound 41 (2,5-bis[2-(5-N-isopropylamidino)benzimidazoyl]pyrrole hydrochloride) is synthesized via condensation of N-isopropylamidino-1,2-phenylene diamine with pyrrole-2,5-dicarboxaldehyde. The reaction proceeds in methanol under reflux, yielding 79% of the target compound with a melting point of 287–289°C.

Table 2: this compound analogues and their synthetic parameters

These analogues retain the bisamidino motif while introducing steric bulk (e.g., cyclopentyl groups) to modulate DNA minor-groove binding.

Pharmaceutical Formulation Methods

Reconstitution for Parenteral Administration

This compound isetionate, the water-soluble salt, requires reconstitution for clinical use. According to the European Medicines Agency , 5 mL of sterile water is added to a 300 mg vial, yielding a 60 mg/mL solution. This solution is further diluted in 50–200 mL of 5% glucose or 0.9% sodium chloride, with gentle inversion to ensure homogeneity.

Table 3: Reconstitution parameters for this compound isetionate

Visual inspection for particulate matter is mandatory, as crystallization under improper storage conditions compromises safety.

Inclusion Complexes for Enhanced Delivery

Recent advances exploit host-guest chemistry to improve this compound’s solubility and targeting. Carboxylated pillararene (CPA5) forms stable inclusion complexes with this compound in three distinct crystalline forms. The primary interaction involves amidinium-carboxylate hydrogen bonds (2.47 Å O–H···O distance), rigidifying the assembly.

Table 4: Crystallographic data for this compound-CPA5 complexes

NMR studies confirm inclusion in solution, with methylene proton broadening (δ 1.2–1.8 ppm) indicating cavity penetration.

Analytical and Characterization Techniques

Spectroscopic Validation

¹H NMR and ¹³C NMR are indispensable for verifying this compound’s structure. For compound 42 , aromatic protons resonate at δ 7.77 (d, J = 8.4 Hz), while cyclopentyl groups exhibit multiplet signals at δ 1.50–2.11 ppm. Mass spectrometry (FAB) corroborates molecular ions, e.g., m/z 520 for compound 42 .

Crystallographic Insights

Single-crystal X-ray diffraction reveals this compound’s extended conformation within CPA5, with host-guest stoichiometries varying by crystallization conditions. Complex III adopts a 2:1 host-guest ratio stabilized by bifurcated H-bonds (2.51 Å), underscoring the role of solvation in polymorph selection.

“The interplay between this compound’s amidinium groups and macrocyclic hosts opens new avenues for controlled drug delivery.”

Types of Reactions: Pentamidine undergoes various chemical reactions, including reduction and substitution reactions. One notable reaction is its electrochemical reduction, which has been studied using cyclic voltammetry .

Common Reagents and Conditions:

Reduction: In a phosphate buffer of pH 8.5, this compound produces a sensitive reduction wave at a potential of -1.56 V (vs.

Substitution: this compound can undergo substitution reactions with nucleophiles, leading to the formation of various derivatives.

Major Products: The major products formed from these reactions include reduced forms of this compound and its various substituted derivatives .

Medical Applications

1.1 Treatment of Pneumocystis Pneumonia

Pentamidine is notably used in the treatment and prophylaxis of Pneumocystis jirovecii pneumonia, particularly in immunocompromised patients, such as those with HIV/AIDS. The drug is administered intravenously or via inhalation.

• Dosage and Administration :
  • Treatment : 3 to 4 mg/kg intravenously daily for 14 to 21 days.
  • Prophylaxis : 300 mg nebulized every four weeks or 4 mg/kg intravenously monthly .

1.2 Leishmaniasis Treatment

This compound is effective against both cutaneous and visceral leishmaniasis, caused by protozoan parasites transmitted through sand fly bites.

• Dosage :
  • Cutaneous Leishmaniasis : 2 to 3 mg/kg daily for four to seven days.
  • Visceral Leishmaniasis : 2 to 4 mg/kg daily for fifteen days or an alternative regimen based on patient response .

1.3 Trypanosomiasis (Sleeping Sickness)

This compound is the first-line treatment for the hemolymphatic stage of Trypanosoma brucei gambiense infection.

• Dosage : 4 mg/kg daily for seven to ten days .

Emerging Applications

2.1 Anticancer Properties

Recent studies have highlighted this compound's potential in oncology, particularly against glioblastomas. Research indicates that this compound can inhibit the proliferation of glioma-initiating cells and induce apoptosis in differentiated glioblastoma cells.

• Mechanism of Action :
  • Suppresses glucose metabolism and RNA synthesis, leading to reduced cell viability and migration .

Case Study: Prophylaxis in Hematopoietic Stem Cell Transplant Patients

A retrospective study evaluated the use of intravenous this compound for Pneumocystis jirovecii pneumonia prophylaxis in patients undergoing hematopoietic stem cell transplantation. The study involved 202 patients who received a median of three doses of intravenous this compound.

• Findings :
  • No cases of breakthrough pneumonia were reported.
  • The treatment was well tolerated with no significant adverse effects noted .

Case Study: Efficacy of Inhaled this compound

A controlled study focused on the efficacy of aerosolized this compound in preventing Pneumocystis pneumonia in HIV-positive patients demonstrated a significant reduction in pneumonia cases compared to placebo (8 vs. 23 cases) with a tolerance rate of about 33% experiencing moderate-to-severe coughing during inhalation .

Data Tables

The exact mechanism of action of pentamidine is not fully understood. it is believed to interfere with nuclear metabolism by inhibiting the synthesis of DNA, RNA, phospholipids, and proteins . This compound binds to nucleic acids through its positively charged amidinium ions, which direct it towards the negatively charged phosphate backbone of DNA and RNA . This interaction disrupts the normal functioning of the nucleic acids, leading to the antimicrobial effects of the drug .

DNA-Binding Diamidines

Berenil (Diminazene Aceturate)

• Structure : Aromatic diamidine with a triazene linker.
• Mechanism : Binds AT-rich DNA similarly to pentamidine but with a preference for longer AT sequences (>4 base pairs) .
• Efficacy: Comparable antiparasitic activity but shares this compound’s toxicity profile. Unlike this compound, it is a substrate for the high-affinity this compound transporter (HAPT1), contributing to cross-resistance in trypanosomes .
• Key Difference : Shorter half-life and higher susceptibility to resistance development .

Furamidine (DB75)

• Structure : Phenyl rings replaced with furan, retaining terminal amidines.
• Mechanism : Binds DNA with higher specificity for AT regions. Its prodrug, DB289 (pafuramidine), is orally bioavailable .
• Efficacy : In Phase III trials for sleeping sickness, pafuramidine showed an 89% cure rate vs. This compound’s 95%, but with superior safety (lower nephrotoxicity) .
• Advantage : Oral administration and reduced cytotoxicity .

Table 1: DNA-Binding Diamidines

Pyridyl Analogs of this compound

Replacement of phenyl rings with pyridyl groups generated 18 analogs ():

• Activity Trends :
  • 2,5-substituted pyridyl dications (e.g., Compound 6) showed superior anti-T. brucei activity (IC50 = 0.001 µM) vs. This compound (IC50 = 0.01–0.1 µM) but poor oral efficacy.
  • N-alkylation reduced cytotoxicity but also anti-parasitic potency.
• Prodrug Strategy : Diamidoxime prodrug (Compound 9) achieved 100% cure in murine models via oral administration, addressing this compound’s bioavailability limitations .

NMDA Receptor Inhibitors

This compound and related diamidines inhibit NMDA receptors via a two-component mechanism:

• Shallow Site Binding : Voltage-independent inhibition (Kvi = 3–30 µM) .
• Deep Site Binding: Voltage-dependent block; this compound, furamidine, and nafamostat exhibit strong trapping block (Kvd = 0.1–3 µM) vs. non-trapping inhibitors like gabexate .

Table 2: NMDA Receptor Inhibition Profiles

Antifungal and Anticancer Derivatives

• Antifungal Bis-Benzimidazoles : Analogs with alkanediamide linkers and heteroatoms (e.g., S, O) showed MIC80 values ≤0.09 µg/mL against Candida albicans, comparable to amphotericin B .
• Anticancer Diamidines : Decamidine and hexamidine outperformed this compound in inhibiting PRMT1, a histone methyltransferase overexpressed in neuroblastoma (cytotoxicity rank: decamidine > hexamidine > this compound) .

Organometallic Complexes

Iridium- and platinum-pentamidine complexes (e.g., cis-Pt(II)-Pentamidine) demonstrated trypanocidal IC50 values of 0.1–2 ng/mL, matching this compound’s efficacy but with 10–20x lower toxicity in murine models .

Pentamidine is a compound that has garnered significant attention for its diverse biological activities, particularly in the fields of oncology and infectious diseases. Originally developed as an antimicrobial agent, its potential applications have expanded due to its ability to interact with various biological targets. This article explores the biological activity of this compound, focusing on its mechanisms of action, therapeutic applications, and recent research findings.

This compound exhibits multiple mechanisms of action that contribute to its biological activity:

• Inhibition of Phosphatases : Research indicates that this compound inhibits PRL phosphatases, which are implicated in cancer progression. This inhibition leads to reduced tumor growth and increased necrosis in cancer cells, particularly in melanoma models .
• T-cell Activation : this compound has been identified as a small-molecule antagonist of PD-L1, enhancing T-cell-mediated cytotoxicity against cancer cells. It promotes the secretion of cytokines such as IFN-γ and TNF-α, thereby restoring T-cell activity and inhibiting tumor growth in vivo .
• DNA Binding : The compound binds to nucleic acids, particularly targeting the minor groove of DNA. This binding is selective for AT-rich regions, which may disrupt essential cellular processes like mitochondrial translation .

Antimicrobial Uses

This compound is primarily known for treating:

• African Trypanosomiasis : Effective against Trypanosoma brucei, the causative agent of sleeping sickness.
• Leishmaniasis : Used in treating various forms of leishmaniasis.
• Pneumocystis Pneumonia : Commonly administered to immunocompromised patients.

Anticancer Potential

Recent studies have highlighted this compound's potential in cancer therapy:

• Prostate Cancer : this compound has shown efficacy in inhibiting prostate cancer cell proliferation and migration. In vivo studies demonstrated significant tumor growth suppression without notable toxicity .
• Melanoma : Inhibition of PRL phosphatases by this compound correlates with reduced tumor size and increased cell death in melanoma models .

Table 1: Summary of this compound's Biological Activities

Recent Research Insights

• Cancer Immunotherapy : A study demonstrated that this compound could serve as a novel PD-L1 antagonist, potentially offering an alternative to monoclonal antibody therapies by enhancing T-cell responses against tumors .
• Combination Therapies : Investigations into drug combinations suggest that this compound can be effectively paired with other agents to enhance efficacy against Trypanosoma cruzi in Chagas disease models .
• Mechanistic Studies : Detailed mechanistic studies have shown that this compound induces mitochondrial dysfunction in cancer cells, leading to apoptosis and reduced cell viability .

Q. What are the critical safety considerations when handling pentamidine in laboratory settings?

Basic Research Question
this compound requires strict safety protocols due to limited toxicity data. Key measures include:

• Engineering controls : Use fume hoods with adequate ventilation and ensure accessible safety showers/eyewash stations.
• PPE : Wear impermeable lab coats, nitrile gloves, side-shielded goggles, and NIOSH-approved respirators when aerosol risk exists .
• Spill management : Avoid drainage contamination; use inert absorbents for cleanup.
• Fire hazards : Employ CO₂ or dry chemical extinguishers; firefighters require SCBA gear due to irritant fumes .

Q. How is the in vitro cytotoxicity of this compound typically assessed, and what parameters are critical for interpreting IC₅₀ values?

Basic Research Question
Standard assays include:

• WST-1 proliferation assays : Seed cells (e.g., HeLa at 10,000 cells/well in 96-well plates) in DMEM + 10% FBS. Treat with this compound (e.g., 0–100 µM) for 24 hr. Measure absorbance post-WST-1 incubation .
• Caspase 3/7 activity assays : Quantify apoptosis via luminescence.
• IC₅₀ interpretation : Report mean ± SD (e.g., 44.16 ± 5.9 µM for HeLa). Ensure consistency in cell density, serum concentration, and exposure duration to minimize variability .

Q. What methodological approaches are recommended for investigating this compound's interaction with structured RNAs like tRNA?

Advanced Research Question

• Native PAGE binding assays : Incubate pure tRNA (e.g., tRNALeu) with this compound (0–400 µM) in varying Mg²⁺ concentrations (0–5 mM). Observe electrophoretic mobility shifts; reduced migration indicates binding .
• pH-dependent studies : Assess binding affinity at pH 5.0–8.0. Electrostatic interactions dominate in low Mg²⁺, while hydrophobic interactions prevail in high Mg²⁺ .
• Functional assays : Couple binding studies with mitochondrial translation inhibition assays (e.g., amino acid misincorporation via radiolabeling) to validate biological impact .

Q. How can researchers design experiments to study the impact of this compound on mitochondrial translation in eukaryotic models?

Advanced Research Question

• Model selection : Use Leishmania mexicana or HeLa cells with intact mitochondrial networks.
• Mitochondrial isolation : Treat cells with this compound (10–50 µM), isolate mitochondria via differential centrifugation, and quantify RNA-drug binding via qPCR or Northern blot .
• Phenotypic assays : Measure oxygen consumption (Seahorse Analyzer) and ATP production to correlate binding with functional deficits .

Q. What strategies are effective in elucidating this compound's inhibitory effects on human enzymes such as diamine oxidase (hDAO)?

Advanced Research Question

• Global nonlinear regression : Fit inhibition curves (e.g., 0–2 µM this compound) to determine Kᵢ values. Use purified hDAO and substrates (e.g., histamine) in kinetic assays .
• Competitive vs. non-competitive analysis : Vary substrate concentrations ± this compound. Lineweaver-Burk plots distinguish inhibition modes .
• Cross-validation : Compare inhibition patterns with structurally similar inhibitors (e.g., berenil) to identify binding site specificity .

Q. How do carbon source variations in growth media influence this compound resistance profiles in bacterial studies?

Advanced Research Question

• Media optimization : Culture Acinetobacter baumannii in M9 minimal media with alternate carbon sources (e.g., glucose vs. succinate).
• Disk diffusion assays : Compare zone-of-inhibition diameters under different conditions. Resistance linked to AdeAB efflux pump activity is carbon-source-dependent .
• Transcriptomic analysis : Perform RNA-seq to identify upregulated transporters (e.g., AdeRS regulon) under selective pressure .

Q. What are the recommended storage conditions for this compound based on its physicochemical properties?

Basic Research Question

• Storage : Keep in airtight containers at 15–25°C, protected from humidity (hygroscopic solid).
• Stability : Limited decomposition data; avoid prolonged exposure to light or temperatures >186°C (melting point) .

Q. What experimental models are suitable for studying this compound's transport mechanisms across the blood-brain barrier (BBB)?

Advanced Research Question

• In vivo models : Use P-gp knockout mice (e.g., mdr1a/mdr1b⁻/⁻) to quantify brain uptake via LC-MS. Wild-type controls assess efflux efficiency .
• In vitro BBB models : Co-culture brain endothelial cells with astrocytes. Apply this compound (1–10 µM) and measure transendothelial electrical resistance (TEER) and permeability .

Q. How can researchers address contradictory findings regarding this compound's primary molecular targets across different experimental systems?

Advanced Research Question

• Systems biology approaches : Integrate proteomics (e.g., affinity purification-MS) and transcriptomics to identify context-dependent targets.
• Dose-response validation : Replicate studies in L. mexicana (mitochondrial target) vs. mammalian cells (tRNA/rRNA) using identical drug concentrations .
• Mechanistic redundancy analysis : Use CRISPR knockouts (e.g., mitochondrial RNA polymerase) to dissect target contributions .

Q. What analytical techniques are employed to validate this compound's purity and stability in experimental preparations?

Basic Research Question

• HPLC-UV/LC-MS : Quantify purity (≥99%) using C18 columns (mobile phase: acetonitrile/0.1% TFA).
• Stability testing : Incubate stock solutions at 4°C/-20°C; monitor degradation via peak area changes over time .